AMPA receptors mediate fast excitatory neurotransmission, contribute to high cognitive processes such as learning and memory and are implicated in numerous psychiatric and neurodegenerative diseases. In particular, AMPA receptors play a key role in epileptogenesis and seizure spread and, thus, have recently emerged as one of the most promising targets for epilepsy therapy. However, development of drugs targeting AMPA receptors has been stalled because of the lack of knowledge about AMPA receptor structure and function. For example, only structures of homotetrameric intact AMPA receptors have been determined, while the overwhelming majority of AMPA receptors in the central nervous system are heterotetramers. A number of noncompetitive inhibitors and ion channel blockers have been identified as promising candidates for drug development but structural mechanisms of their action on AMPA receptors remain largely unexplored. This missing information is absolutely critical for the future structure-based rational drug design. We plan to study structure and function of AMPA receptors using a combination of biophysical and biochemical approaches, including modern crystallographic and cryo-electron microscopy (cryo-EM) techniques, fluorescence-based methods, electrophysiology, kinetic and molecular modeling. Our specific aims are to (1) obtain structures of heteromeric AMPA receptors, (2) establish the molecular mechanism of noncompetitive inhibition, and (3) build a structural model of ion channel block. To reach our goals, we will optimize AMPA receptor constructs for crystallization and cryo-EM experiments, develop protocols of their expression and purification and solve structures of heterotetrameric AMPA receptors and AMPA receptors in complex with noncompetitive inhibitors and ion channel blockers. To improve our structural models, we will use new methods of structural refinement combined with molecular dynamics (MD) simulations. We will also test our models using a combination of experimental and in silico mutagenesis, whole-cell patch-clamp recordings and MD simulations. To understand the molecular mechanisms of AMPA receptor heteromeric assembly, noncompetitive inhibition and ion channel block, we will perform extensive MD simulations of homo- and heteromeric AMPA receptors in different activation states and in the presence or absence of noncompetitive inhibitors and ion channel blockers. We will combine the results of structural, computational, functional and mutagenesis experiments to propose molecular models of AMPA receptor heteromeric assembly, noncompetitive inhibition and ion channel block. Reaching our research goals will provide molecular level knowledge essential to greatly facilitate design of new molecules that will have a potential to become safe and more efficacious drugs to treat epilepsy and other disorders related to excitatory neurotransmission.